US20040019018A1 - Rigid pyrrolidone modulators of pkc - Google Patents

Rigid pyrrolidone modulators of pkc Download PDF

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US20040019018A1
US20040019018A1 US10/258,661 US25866103A US2004019018A1 US 20040019018 A1 US20040019018 A1 US 20040019018A1 US 25866103 A US25866103 A US 25866103A US 2004019018 A1 US2004019018 A1 US 2004019018A1
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pkc
compound
cycloalkyl
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aryl
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Lixin Qlao
Alan Kozikowski
Lianyun Zhao
Rene Etcheberrigaray
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D209/00—Heterocyclic compounds containing five-membered rings, condensed with other rings, with one nitrogen atom as the only ring hetero atom
    • C07D209/56—Ring systems containing three or more rings
    • C07D209/58—[b]- or [c]-condensed
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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Definitions

  • This invention relates to molecules that modulate the biological activities of isozymes of protein kinase C (PKC), thereby affecting diverse cellular functions, including cell growth, cell differentiation, apoptosis, ion channel activity, neurotransmitter release, and neuronal plasticity.
  • PKC protein kinase C
  • the invention relates to tricyclic, rigid pyrrolidone compounds and derivatives thereof that bind to PKC, comprise a tricyclic structure of fused pyrrole, cycloalkyl, and phenyl rings, and mimic the binding of naturally occurring modulators of PKC activity such as diacylglycerol and indolactam V (ILV).
  • the compounds of the present invention may be advantageously used, for example, in the treatment of cancerous, autoimmune, inflammatory and neurologic diseases, and for the treatment of conditions associated with amyloid processing and plaque formation.
  • PKC protein kinase C
  • the PKC gene family consists presently of 11 genes which are divided into four subgroups: 1) classical PKC ⁇ , ⁇ 1 , ⁇ 2 ( ⁇ 1 and ⁇ 2 are alternately spliced forms of the same gene) and ⁇ ; 2) novel PKC delta ( ⁇ ), epsilon ( ⁇ ), eta ( ⁇ ), and theta ( ⁇ ); 3) atypical PKC zeta ( ⁇ ), lambda ( ⁇ ) and iota ( ⁇ ); and 4) PKC ⁇ .
  • PKC ⁇ resembles the novel PKC isozymes but differs by having a putative transmembrane domain (reviewed in Blobe, et al., Cancer Metast. Rev., 13, 411 (1994)).
  • the ⁇ , ⁇ 1 , ⁇ 2 and ⁇ isozymes are Ca 2+ , phospholipid- and diacylglycerol-dependent and represent the classical isozymes of PKC, whereas the other isozymes are activated by phospholipid and diacylglycerol but are not dependent on Ca 2+ . All isozymes encompass 5 variable (V1-V5) regions, and the alpha, beta and gamma isozymes contain four (C1-C4) structural domains which are highly conserved.
  • All isozymes except PKC alpha, beta, and gamma lack the C2 domain, and the lambda, eta and iota isozymes also lack one of two cysteine-rich zinc finger domains in C1 to which diacylglycerol binds.
  • the C1 domain also contains the pseudosubstrate sequence which is highly conserved among all isozymes, and which serves an autoregulatory function by blocking the substrate-binding site to produce an inactive conformation of the enzyme (House et al. Science, 238, 1726 (1987)).
  • PKC has served as a focal point for the design of anticancer drugs (Gescher, Brit. J. Cancer, 66, 10 (1992)).
  • Antisense expression of either the PKC ⁇ cDNA (Ahmad, et al., Neurosurgery, 35, 904 (1994)) or a phosphorothioate oligodeoxynucleotide (S-oligo) for PKC alpha has shown the efficacy of targeting PKC to inhibit the proliferation of A549 lung carcinoma cells (Dean, et al., J. Biol. Chem., 269, 16416 (1994)) and U-87 glioblastoma cells.
  • isozymes are most crucial for tumor proliferation and what specific roles different PKC isozymes play in critical cellular processes such as cell proliferation and apoptosis.
  • Prostate cancer is a leading cause of cancer death among men in Western countries.
  • PKC activator 12-O-tetradecanoyl-phorbol-13-acetate (TPA) promotes cell death in androgen-sensitive LNCaP cells, rather than androgen-independent DU-145 or PC-3 cells, whose growth is significantly decreased by PKC inhibitor staurosporine.
  • PKC ⁇ is therefore an attractive and novel target for the therapy of androgen-independent prostate cancer, because PKC isozymes are involved in the regulation of prostate cancer cell growth and apoptosis.
  • TPA tumor promoters
  • PKC membrane-associated high affinity receptors
  • the present invention provides certain substituted polycylic, rigid pyrrolidones that are PKC modulators. Accordingly, the invention provides a compound of the general structural formula (I):
  • the compounds comprise a pyrrole ring fused to a first cyclic substituent (B), wherein the first cyclic substituent is also fused to a second cyclic substituent (A).
  • the second cyclic substituent is preferably a carbocyclic ring, preferably a C 5 -C 7 cycloalkyl group and more preferably a phenyl ring.
  • R 1 , R 2 , and R 3 are each independently hydrogen, (C 1 -C 15 )alkyl, (C 2 -C 15 )alkenyl, (C 2 -C 15 )alkynyl, (C 3 -C 8 )cycloalkyl, (C 3 -C 8 )cycloalkyl(C 1 -C 10 )alkyl, (C 3 -C 8 )cycloalkyl(C 1 -C 15 )alkenyl, (C 3 -C 8 )cycloalkyl(C 1 -C 15 )alkynyl, (C 1 -C 15 )alkoxy, (C 1 -C 15 )alkanoyl, (C 1 -C 15 )alkanoyloxy, aryl heteroaryl, aryl(C 1 -C 6 )alkyl, heteroaryl(C 1 -C 1 )alkyl, aryl heteroaryl, aryl(C 1
  • R 4 comprises 0-4 substituents, independently selected from the group consisting of halo, nitro, cyano, hydroxy, phospho, sulfo, trifluoromethyl, trifluoromethoxy, (C 1 -C 15 )alkyl, (C 2 -C 15 )alkenyl, (C 2 -C 15 )alkynyl, (C 3 -C 8 )cycloalkyl, (C 3 -C 8 )cycloalkyl(C 1 -C 15 )alkyl, (C 3 -C 8 )cycloalkyl(C 2 -C 15 )alkenyl, (C 3 -C 8 )cycloalkyl(C 2 -C 15 )alkynyl, (C 1 -C 15 )alkoxy, (C 1 -C 15 )alkanoyl, (C 1 -C 15 )alkanoyloxy, C( ⁇ O)OR a , C(
  • Preferred embodiments include compounds of Formula I and II, with the substituents as described above.
  • the invention also provides pharmaceutical compositions comprising a compound of the invention, processes for preparing compounds of the invention, and novel intermediates useful for the synthesis of compounds of the invention.
  • the invention also provides a therapeutic method comprising treating a condition characterized by the pathological proliferation of mammalian cells (e.g. cancers and tumors, and in a preferred embodiment, prostate cancer) by administering to a mammal afflicted with such a condition an effective amount of a substituted tricyclic, rigid pyrrolidone with a pharmaceutically acceptable carrier.
  • mammalian cells e.g. cancers and tumors, and in a preferred embodiment, prostate cancer
  • the invention also provides a method comprising modulating PKC activity in a mammal by administering to said mammal an effective dose of a substituted tricyclic, rigid pyrrolidone with a pharmaceutically acceptable carrier.
  • the invention provides a method of treating a patient having an autoimmune disease, by administering to said mammal an effective dose of a substituted tricyclic, rigid pyrrolidone with a pharmaceutically acceptable carrier.
  • the invention provides a method of treating a patient having an inflammation, by administering to said mammal an effective dose of a substituted tricyclic, rigid pyrrolidone with a pharmaceutically acceptable carrier.
  • the present invention also relates to the use of rigid pyrrolidones to alter conditions associated with amyloid processing in order to enhance an ⁇ -secretase pathway to generate soluble ⁇ -amyloid precursor protein ( ⁇ -APP) so as to reduce or prevent ⁇ -amyloid aggregation including ⁇ -amyloid accumulation in neurons.
  • ⁇ -APP soluble ⁇ -amyloid precursor protein
  • Such activation for example, can be employed in the treatment of Alzheimer's disease, neurological disease, senile plaques, or cerebral amyloid angiopathy (CAA).
  • CAA cerebral amyloid angiopathy
  • the present invention relates to a method for treating plaque formation, such as that associated with Alzheimer's disease, senile plaques, or CAA comprising administering to a subject an effective amount of a rigid pyrrolidone.
  • the invention is also a method of modulating K + channel conductance by administering an effective amount of a pharmaceutical composition comprising a rigid pyrrolidone.
  • the present invention relates to a method for altering conditions associated with amyloid processing in order to enhance an ⁇ -secretase pathway to generate soluble ⁇ -amyloid precursor protein ( ⁇ -APP) so as to prevent ⁇ -amyloid aggregation comprising administering a biologically effective amount of a rigid pyrrolidone.
  • ⁇ -APP soluble ⁇ -amyloid precursor protein
  • the present invention relates to a composition for treating plaque formation, such as that associated with Alzheimer's disease comprising: a rigid pyrrolidone in an amount effective to generate soluble ⁇ -APP and reduce or prevent ⁇ -amyloid aggregation; and a pharmaceutically acceptable carrier.
  • isozyme selective, non-tumor promoting activators of PKC may find use in cancer treatment through the initiation of cancer cell death by apoptosis. Selective cancer cell killing may be achieved either through the targeting of those isozymes found to be overexpressed in the cancer cells, or through the synergistic interaction of a cytotoxic drug like 1-beta-D-arabinofuranosylcytosine with an appropriate PKC-based signaling interceptor.
  • compounds of the invention may also be useful as pharmacological tools for the in vitro or in vivo study of the physiological function and effects of the PKC gene family.
  • the substituted tricyclic, rigid pyrrolidone compounds of the present invention exhibit distinct advantages over the prior art. These compounds have merit for several reasons, including availability, diversity, inexpensive starting material, and simplicity of the overall synthesis.
  • FIG. 1 illustrates the overall features of the binding model of a compound of the invention and a benzolactam in complex with PKC ⁇ C1B.
  • FIG. 2 illustrates a synthetic reaction pathway for synthesis of a substituted tricyclic, rigid pyrrolidone of the present invention, (3S, 8R, 9S, 10S)-6-(dec-1′-ynyl)-3-hydroxymethyl-8-isopropyl-8-methyl-3,3a,8,8-tetrahydro-ZH-aza-cyclopenta[a]inden-1-one.
  • FIG. 3 illustrates synthetic reaction pathways for some di- and tri-substituted compounds of the invention.
  • FIG. 4 is a schematic representation of the various stages in the calcium signaling cascade.
  • FIG. 5 shows a western blot demonstrating the effect of compound 1 of the invention on sAPP ⁇ .
  • FIG. 6 is a graph of the induction of apoptosis in LNCaP prostate cancer cells by compound (1) or PMA.
  • the front methyl group on the marked chiral center mimics the N-Me in BL, interacting with Leu250 of PKC.
  • the rear isopropyl group of compound (1) interacts with side chain of Leu254, thereby mimicking the isopropyl group of BL.
  • the same orientation of the phenyl group in compound (1) allows for strong hydrophobic interactions with Pro241 of PKC.
  • Halo is fluoro, chloro, bromo, or iodo.
  • Alkyl, alkoxy, etc. denote both straight and branched groups; but reference to an individual radical such as “propyl” embraces only the straight chain radical, a branched chain isomer such as “isopropyl” being specifically referred to.
  • Aryl denotes a phenylradical or an ortho-fused bicyclic carbocyclic radical having about nine to ten ring atoms in which at least one ring is aromatic.
  • Heteroaryl encompasses a radical attached via a ring carbon of a monocyclic aromatic ring containing five or six ring atoms consisting of carbon and one to four heteroatoms each selected from the group consisting of non-peroxide oxygen, sulfur, and N(R x ) wherein R x is absent or is hydrogen, oxo, (C 1 -C 4 )alkyl, phenyl or benzyl, as well as a radical of an ortho-fused bicyclic heterocycle of about eight to ten ring atoms derived therefrom, particularly a benzo-derivative or one derived by fusing a propylene, trimethylene, or tetramethylene diradical thereto.
  • cyclic substituent herein denotes any aryl, heteroaryl, aromatic, cycloalkyl, or other cyclic moiety containing at least one continuous, cyclic pathway of covalently bonded constituent atoms.
  • fused denotes a covalent connection between two cyclic substituents of a molecule such that at least two adjacent atoms are common to both ring structures, and no independent rotational degree of freedom exists between the two cyclic substituents.
  • tricyclic, rigid pyrrolidone herein denotes a pyrrolidone of Formula III comprising a pyrrole ring fused to a first cyclic substituent (B), wherein the first cyclic substituent is also fused to a second cyclic substituent (A).
  • the second cyclic substituent is preferably a carbocyclic ring, preferably a C 5 -C 7 cycloalkyl group and more preferably a phenyl ring.
  • the second cyclic substituent is may be substituted with one or more groups R 4 .
  • Compounds having a substituted or unsubstituted phenyl ring as the second cyclic substituent (Formula I) and a separate spirocyclic substituent as R 1 and R 2 (Formula II) are preferred tricyclic, rigid pyrrolidones.
  • R 1 , R 2 or R 3 a specific value for R 1 , R 2 or R 3 , is (C 1 -C 15 )alkyl, (C 2 -C 15 )alkenyl, C 2 -C 15 )alkynyl, (C 3 -C 8 ) cycloalkyl, (C 3 -C 8 ) cycloalkyl(C 1 -C 10 )alkyl, (C 3 -C 8 )cycloalkyl(C 1 -C 15 )alkenyl, (C 3 -C 8 )cycloalkyl(C 1 -C 15 )alkynyl, (C 1 -C 15 )alkoxy, (C 1 -C 15 )alkanoyl, or (C 1 -C 15 )alkanoyloxy; wherein R 1 is optionally substituted with one or more (e.g.
  • substituents independently selected from the group consisting of halo, nitro, cyano, hydroxy, trifluoromethyl, trifluoromethoxy, (C 1 -C 15 )alkyl, (C 2 -C 15 )alkenyl, (C 2 -C 15 )alkynyl, (C 3 -C 8 )cycloalkyl, (C 3 -C 8 )cycloalkyl (C 1 -C 15 )alkyl, (C 3 -C 8 )cycloalkyl-(C 2 -C 15 )alkenyl, (C 3 -C 8 )cycloalkyl(C 2 -C 15 )alkynyl, (C 1 -C 15 )alkoxy, (C 1 -C 15 )alkanoyl, (C 1 -C 15 )alkanoyloxy, C( ⁇ O)OR a , C( ⁇ O)NR b R c , OC
  • R 1 , R 2 or R 3 Another specific value for R 1 , R 2 or R 3 , is (C 1 -C 15 )alkyl, (C 2 -C 15 )alkenyl, (C 2 -C 15 )alkynyl, (C 3 -C 8 )cycloalkyl, (C 3 -C 8 )cycloalkyl(C 1 -C 10 )alkyl, (C 3 -C 8 )cycloalkyl(C 1 -C 15 )alkenyl, C 3 -C 8 )cycloalkyl(C 1 -C 15 )alkynyl, (C 1 -C 15 )alkoxy, (C 1 -C 15 )alkanoyl, or (C 1 -C 15 )alkanoyloxy; wherein R 1 is optionally substituted with one or more (e.g.
  • substituents independently selected from the group consisting of halo, nitro, cyano, hydroxy, trifluoromethyl, trifluoromethoxy, C( ⁇ O)OR a , C( ⁇ O)NR b R c , OC( ⁇ O)OR a , OC( ⁇ O)NR b R c , and NR e R f .
  • R 1 , R 2 or R 3 is aryl optionally substituted with 1, 2, or 3 substituents independently selected from the group consisting of halo, nitro, cyano, hydroxy, trifluoromethyl, trifluoromethoxy, (C 1 -C 6 )alkyl, (C 2 -C 10 )alkenyl, (C 2 -C 10 )alkynyl, (C 3 -C 8 )cycloalkyl, (C 3 -C 8 )cycloalkyl(C 1 -C 6 )alkyl, (C 1 -C 10 )alkoxy, (C 1 -C 10 )alkanoyl, (C 2 -C 10 )alkanoyloxy, C( ⁇ O)OR a , C( ⁇ O)NR b R c , or NR e R f .
  • substituents independently selected from the group consisting of halo, nitro, cyano, hydroxy, trifluoromethyl,
  • R 1 , R 2 or R 3 is phenyl or naphthyl, optionally substituted with 1, 2, or 3 substituents independently selected from the group consisting of halo, nitro, cyano, hydroxy, trifluoromethyl, trifluoromethoxy, (C 1 -C 6 )alkyl, (C 2 -C 10 )alkenyl, (C 2 -C 10 ) alkynyl, (C 3 -C 8 )cycloalkyl, (C 3 -C 8 )cycloalkyl(C 1 -C 6 )alkyl, (C 1 -C 10 )alkoxy, (C 1 -C 10 )alkanoyl, (C 2 -C 10 )alkanoyloxy, C( ⁇ O)OR a C( ⁇ O)NR b R c , or NR e R f .
  • substituents independently selected from the group consisting of halo, nitro, cyano, hydroxy,
  • R 1 , R 2 or R 3 is phenyl or naphthyl, optionally substituted with a substituent selected from the group consisting of halo, nitro, cyano, hydroxy, trifluoromethyl, trifluoromethoxy, (C 1 -C 6 )alkyl, (C 2 -C 10 )alkenyl, (C 2 -C 10 )alkynyl, (C 3 -C 8 )cycloalkyl, (C 3 -C 8 )cycloalkyl(C 1 -C 6 )alkyl, (C 1 -C 10 alkoxy, (C 1 -C 10 )alkanoyl, (C 2 -C 10 )alkanoyloxy, C( ⁇ O)OR a , C( ⁇ O)NR b R c , or NR e R f .
  • a substituent selected from the group consisting of halo, nitro, cyano, hydroxy, trifluoro
  • R 1 , R 2 or R 3 Another specific value for R 1 , R 2 or R 3 , is aryl, heteroaryl, aryl(C 1 -C 6 )alkyl, heteroaryl(C 1 -C 6 )alkyl, aryl(C 2 -C 6 )alkenyl, heteroaryl(C 2 -C 6 )alkenyl, aryl(C 2 -C 6 )alkynyl, or heteroaryl(C 2 -C 6 )alkynyl; wherein any aryl or heteroaryl of R 1 is optionally substituted with one or more (e.g.
  • substituents independently selected from the group consisting of halo, nitro, cyano, hydroxy, trifluoromethyl, trifluoromethoxy, (C 1 -C 15 )alkyl, (C 2 -C 15 )alkenyl, (C 2 -C 15 )alkynyl, (C 3 -C 8 )cycloalkyl, (C 3 -C 8 )cycloalkyl(C 1 -C 15 )alkyl, (C 3 -C 8 )cycloalkyl-(C 2 -C 15 )alkenyl, (C 3 -C 8 )cycloalkyl(C 2 -C 15 )alkynyl, (C 1 -C 15 )alkoxy, (C 1 -C 15 )alkanoyl, (C 1 -C 15 )alkanoyloxy, C( ⁇ O)OR a , C( ⁇ O)NR b R c , OC
  • R 1 , R 2 or R 3 Another specific value for R 1 , R 2 or R 3 , is aryl, heteroaryl, aryl(C 1 -C 6 )alkyl, heteroaryl(C 1 -C 6 )alkyl, aryl(C 2 -C 6 )alkenyl, heteroaryl(C 2 -C 6 )alkenyl, aryl(C 2 -C 6 )alkynyl, or heteroaryl(C 2 -C 6 )alkynyl; wherein any aryl or heteroaryl of R 1 is optionally substituted with halo, nitro, cyano, hydroxy, trifluoromethyl, trifluoromethoxy, (C 1 -C 15 )alkyl, (C 2 -C 15 )alkenyl, (C 2 -C 15 )alkynyl, (C 3 -C 8 )cycloalkyl, (C 3 -C 8 )cycloalkyl(
  • R 1 , R 2 or R 3 is aryl or heteroaryl wherein said aryl or heteroaryl is optionally substituted with one or more (e.g. 1, 2, 3, or 4) substituents independently selected from the group consisting of halo, nitro, cyano, hydroxy, trifluoromethyl, trifluoromethoxy, (C 1 -C 15 )alkyl, C 2 -C 15 )alkenyl, (C 2 -C 15 )alkynyl, (C 3 -C 8 )cycloalkyl, (C 3 -C 8 )cycloalkyl(C 1 -C 15 )alkyl, (C 3 -C 8 )cycloalkyl-(C 2 -C 15 )alkenyl, (C 3 -C 8 )cycloalkyl-(C 2 -C 15 )alkenyl, (C 3 -C 8 )cycloalkyl-(C 2 -C 15 )alkenyl
  • R 1 , R 2 or R 3 is aryl or heteroaryl, wherein said aryl or heteroaryl is optionally substituted with halo, nitro, cyano, hydroxy, trifluoromethyl, trifluoromethoxy, (C 1 -C 15 )alkyl, (C 2 -C 15 )alkenyl, (C 2 -C 15 )alkynyl, (C 3 -C 8 )cycloalkyl, (C 3 -C 8 )cycloalkyl(C 1 -C 15 )alkyl, (C 3 -C 8 )cycloalkyl-(C 2 -C 15 )alkenyl, (C 3 -C 8 )cycloalkyl(C 2 -C 15 )alkynyl, (C 1 -C 15 )alkoxy, (C 1 -C 15 )alkanoyl, (C 1 -C 15 )alkanoyl, (C 1 -C 15
  • R 1 , R 2 or R 3 is aryl wherein said aryl is optionally substituted with one or more (e.g. 1, 2, 3, or 4) substituents independently selected from the group consisting of halo, nitro, cyano, hydroxy, trifluoromethyl, trifluoromethoxy, (C 1 -C 15 )alkyl, (C 2 -C 15 )alkenyl, (C 2 -C 15 )alkynyl, (C 3 -C 8 )cycloalkyl, (C 3 -C 8 )cycloalkyl(C 1 -C 15 )alkyl, (C 3 -C 8 )cycloalkyl-(C 2 -C 15 )alkenyl, (C 3 -C 8 )cycloalkyl(C 2 -C 15 )alkynyl, (C 1 -C 15 )alkoxy, (C 1 -C 15 )alkanoy
  • R 1 , R 2 or R 3 is aryl wherein said aryl is optionally substituted with halo, nitro, cyano, hydroxy, trifluoromethyl, trifluoromethoxy, (C 1 -C 15 )alkyl, (C 2 -C 15 )alkenyl, (C 2 -C 15 )alkynyl, (C 3 -C 8 )cycloalkyl, (C 3 -C 8 )cycloalkyl(C 1 -C 15 )alkyl, (C 3 -C 8 )cycloalkyl-(C 2 -C 15 )alkenyl, (C 3 -C 8 )cycloalkyl(C 2 -C 15 )alkynyl, (C 1 -C 15 )alkoxy, (C 1 -C 15 )alkanoyl, (C 1 -C 15 )alkanoyloxy, C( ⁇ O)OR
  • R 1 , R 2 or R 3 is aryl or heteroaryl wherein said aryl or heteroaryl is substituted with one or more (e.g. 1, 2, 3, or 4) substituents independently selected from the group consisting of halo, nitro, cyano, hydroxy, trifluoromethyl, trifluoromethoxy, (C 3 -C 8 )cycloalkyl, (C 3 -C 8 )cycloalkyl(C 1 -C 15 )alkyl, (C 3 -C 8 )cycloalkyl(C 2 -C 15 )alkenyl, (C 3 -C 8 )cycloalkyl(C 2 -C 15 )alkynyl, (C 1 -C 15 )alkoxy, (C 1 -C 15 )alkanoyl, (C 1 -C 15 )alkanoyloxy, C( ⁇ O)OR a , C( ⁇ O)NR b R
  • R 1 , R 2 or R 3 is phenyl or naphthyl, wherein said phenyl or naphthyl is optionally substituted with one or more (e.g. 1, 2, 3, or 4) substituents independently selected from the group consisting of halo, nitro, cyano, hydroxy, trifluoromethyl, trifluoromethoxy, (C 1 -C 6 )alkyl, (C 2 -C 6 )alkenyl, (C 2 -C 6 )alkynyl, (C 3 -C 8 )cycloalkyl, (C 3 -C 8 )cycloalkyl(C 1 -C 6 )alkyl, (C 3 -C 8 )cycloalkyl(C 1 -C 6 )alkenyl, (C 3 -C 8 )cycloalkyl(C 1 -C 6 )alkynyl, (C 1 -C 6 )alkoxy
  • R 1 , R 2 or R 3 Another specific value for R 1 , R 2 or R 3 , is phenyl or naphthyl, wherein said phenyl or naphthyl is optionally substituted with halo, nitro, cyano, hydroxy, trifluoromethyl, trifluoromethoxy, (C 1 -C 15 )alkyl, (C 2 -C 15 )alkenyl, (C 3 -C 8 )cycloalkyl(C 1 -C 15 )alkyl, (C 3 -C 8 )cycloalkyl-(C 2 -C 15 )alkenyl, (C 3 -C 8 )cycloalkyl(C 2 -C 15 )alkynyl, (C 1 -C 15 )alkoxy, (C 1 -C 15 )alkanoyl, (C 1 -C 15 )alkanoyloxy, C( ⁇ O)OR a , C( ⁇ O)NR
  • R 1 , R 2 or R 3 is aryl or heteroaryl, wherein said aryl or heteroaryl is substituted with (C 1 -C 15 )alkyl, (C 2 -C 15 )alkenyl, (C 2 -C 15 )alkynyl, (C 1 -C 15 )alkoxy, (C 1 -C 15 )alkanoyl, or (C 1 -C 15 )alkanoyloxy, and can also be optionally substituted with 1 or 2 halo, nitro, cyano, hydroxy, trifluoromethyl, trifluoromethoxy, (C 1 -C 6 )alkyl, (C 3 -C 8 )cycloalkyl, (C 1 -C 6 )alkoxy, (C 1 -C 6 )alkanoyl, (C 2 -C 6 )alkanoyloxy, C( ⁇ O)OR a , C( ⁇ O)
  • R 1 Another specific value for R 1 is phenyl or naphthyl, wherein said phenyl or naphthyl is substituted (preferably at the 4-position) with (C 1 -C 15 )alkyl, (C 2 -C 15 )alkenyl, (C 2 -C 15 )alkynyl (C 1 -C 15 )alkoxy, (C 1 -C 15 )alkanoyl, or (C 1 -C 15 )alkanoyloxy, and can also be optionally substituted with 1 or 2 halo, nitro, cyano, hydroxy, trifluoromethyl, trifluoromethoxy, (C 1 -C 6 )alkyl, (C 3 -C 8 )cycloalkyl, (C 1 -C 6 )alkoxy, (C 1 -C 6 )alkanoyl, (C 2 -C 6 )alkanoyloxy, C( ⁇ O)OR a , C(C
  • R 1 , R 2 or R 3 is phenyl or naphthyl; optionally substituted with 1, 2, or 3 substituents independently selected from the group consisting of halo, nitro, cyano, hydroxy, trifluoromethyl, trifluoromethoxy, (C 1 -C 6 )alkyl, (C 2 -C 10 )alkenyl, (C 2 -C 10 )alkynyl(C 3 -C 8 )cycloalkyl, (C 3 -C 8 )cycloalkyl(C 1 -C 6 )alkyl, (C 1 -C 10 )alkoxy, C 1 -C 10 )alkanoyl, (C 2 -C 10 )alkanoyloxy, C( ⁇ O)OR a , C( ⁇ O)NR b R c , or NR e R f ; R a is hydrogen or (C 1 -C 6 )alkyl, (C 2 -C 10
  • R 1 , R 2 or R 3 is phenyl or naphthyl, wherein said phenyl or naphthyl is substituted (preferably at the 4-position) with (C 1 -C 15 )alkyl, (C 2 -C 15 )alkenyl, (C 2 -C 15 )alkynyl (C 1 -C 15 )alkoxy, (C 1 -C 15 )alkanoyl, or (C 1 -C 15 )alkanoyloxy.
  • R 1 , R 2 or R 3 Another more specific value for R 1 , R 2 or R 3 , is phenyl or naphthyl, wherein said phenyl or naphthyl is substituted (preferably at the 4-position with (C 1 -C 15 )alkyl, (C 2 -C 15 )alkenyl, or (C 2 -C 15 )alkynyl.
  • R 1 , R 2 or R 3 Another more specific value for R 1 , R 2 or R 3 , is phenyl substituted (preferably at the 4-position) with (C 8 -C 15 )alkyl, (C 8 -C 15 )alkenyl, (C 8 -C 15 )alkynyl, (C 8 -C 15 )alkoxy, (C 8 -C 15 )alkanoyl, or (C 8 -C 15 )alkanoyloxy.
  • R 1 , R 2 or R 3 Another more specific value for R 1 , R 2 or R 3 , is aryl (e.g. phenyl, naphthyl, or 5, 6, 7, 8-tetrahydronaphthyl) substituted (preferably at the 4-position) with (C 7 -C 10 )alkyl, (C 7 -C 10 ) alkenyl, C 7 -C 10 )alkynyl, (C 7 -C 10 )alkoxy, (C 7 -C 10 )alkanoyl, or (C 7 -C 10 )alkanoyloxy.
  • aryl e.g. phenyl, naphthyl, or 5, 6, 7, 8-tetrahydronaphthyl substituted (preferably at the 4-position) with (C 7 -C 10 )alkyl, (C 7 -C 10 ) alkenyl, C 7 -C 10 )alkynyl, (C 7 -C
  • R 1 is naphthyl, optionally substituted (preferably at the 4-position) with (C 1 -C 15 )alkyl, (C 2 -C 15 )alkenyl, (C 2 -C 15 )alkynyl, (C 1 -C 15 )alkoxy, (C 1 -C 15 )alkanoyl, or (C 1 -C 15 )alkanoyloxy.
  • R 1 , R 2 or R 3 Another more specific value for R 1 , R 2 or R 3 , is 5, 6, 7, 8-tetrahydronaphthyl, optionally substituted with (C 1 -C 15 )alkyl, (C 2 -C 15 )alkenyl, (C 2 -C 15 )alkynyl, (C 1 -C 15 )alkoxy, (C 1 -C 15 )alkanoyl, or (C 1 -C 15 )alkanoyloxy, and optionally substituted at the 5, 6, 7, or 8 position with a divalent (C 2 -C 7 )alkylene chain to form a (C 3 -C 8 )spirocycloalkyl.
  • a preferred value for R 1 , R 2 or R 3 is 4-nonylphenyl, phenyl, 1-naphthyl, 4-hexanoyloxynaphth-1-yl, 4-nonanoyloxynaphth-1-yl, 4-(1-hexynyl)naphth-1-yl, 7,7-dimethyl-4-nonanoyloxy-5,6,7,8-tetrahydronaphth-1-yl, 4-nonanoyloxy-5,6,7,8-tetrahydronaphth-1-yl, 4-hexanoyloxy-5,6,7,8tetrahydronaphth-1-yl, 4-nonanoyloxy-7-spirocyclopropyl-5,6,7,8-tetrahydronaphth-1-yl, or 3-pentyl.
  • R 1 , R 2 or R 3 is (C 1 -C 10 )alkyl, (C 2 -C 10 )alkenyl, (C 3 -C 8 )cycloalkyl, (C 3 -C 8 )cycloalkyl(C 1 -C 6 )alkyl, (C 1 -C 10 )alkoxy, (C 1 -C 10 )alkanoyl, or (C 1 -C 10 )alkanoyloxy, optionally substituted with 1, 2, or 3 substituents independently selected from the group consisting of halo, nitro, cyano, hydroxy, trifluoromethyl, trifluoromethoxy, (C 1 -C 6 )alkyl, (C 2 -C 10 )alkenyl, (C 2 -C 10 )alkynyl, (C 3 -C 8 )cycloalkyl, (C 3 -C 8 )cycloalkyl(C 1
  • R 1 , R 2 or R 3 Another specific value for R 1 , R 2 or R 3 , is (C 1 -C 10 )alkyl, (C 2 -C 10 )alkenyl, (C 3 -C 8 )cycloalkyl, (C 3 -C 8 )cycloalkyl(C 1 -C 6 )alkyl, (C 1 -C 10 )alkoxy, (C 1 -C 10 )alkanoyl, or (C 1 -C 10 )alkanoyloxy, optionally substituted with a substituent selected from the group consisting of halo, nitro, cyano, hydroxy, trifluoromethyl, trifluoromethoxy, (C 1 -C 6 )alkyl, (C 2 -C 10 )alkenyl, (C 2 -C 10 )alkynyl, (C 3 -C 8 )cycloalkyl, (C 3 -C 8 )cycloalkyl(
  • R 1 , R 2 or R 3 is (C 1 -C 10 )alkyl, optionally substituted with 1, 2, or 3 substituents independently selected from the group consisting of halo, nitro, cyano, hydroxy, trifluoromethyl, trifluoromethoxy, (C 1 -C 6 )alkyl, (C 2 -C 10 )alkenyl, (C 2 -C 10 )alkynyl, (C 3 -C 8 )cycloalkyl, (C 3 -C 8 )cycloalkyl(C 1 -C 6 )alkyl, (C 1 -C 10 )alkoxy, (C 1 -C 10 )alkanoyl, (C 2 -C 10 )alkanoyloxy, C( ⁇ O)OR a , C( ⁇ O)NR b R c , or NR e R f .
  • substituents independently selected from the group consisting of halo, nitro, cyano
  • R 1 , R 2 or R 3 is (C 1 -C 10 )alkyl, optionally substituted with a substituent selected from the group consisting of halo, nitro, cyano, hydroxy, trifluoromethyl, trifluoromethoxy, (C 1 -C 6 )alkyl, (C 2 -C 10 )alkenyl, (C 2 -C 10 )alkynyl, (C 3 -C 8 ) cycloalkyl, (C 3 -C 8 )cycloalkyl(C 1 -C 6 )alkyl, (C 1 -C 10 )alkoxy, (C 1 -C 10 )alkanoyl, (C 2 -C 10 )alkanoyloxy, C( ⁇ O)OR a , C( ⁇ O)NR b R c , or NR e R f .
  • a substituent selected from the group consisting of halo, nitro, cyano, hydroxy
  • R 1 , R 2 or R 3 Another specific value for R 1 , R 2 or R 3 , is (C 1 -C 15 )alkyl, (C 2 -C 15 )alkenyl, (C 2 -C 15 )alkynyl, (C 3 -C 8 ) cycloalkyl, (C 3 -C 8 )cycloalkyl(C 1 -C 10 )alkyl, (C 1 -C 15 )alkoxy, (C 1 -C 15 )alkanoyl, (C 1 -C 15 )alkanoyloxy, aryl, heteroaryl, aryl(C 1 -C 6 )alkyl, heteroaryl (C 1 -C 6 )alkyl, aryl(C 2 -C 15 )alkenyl, heteroaryl(C 2 -C 15 )alkenyl, aryl(C 2 -C 15 )alkenyl, aryl(C 2 -C
  • substituents independently selected from the group consisting of halo, nitro, cyano, hydroxy, trifluoromethyl, trifluoromethoxy, (C 1 -C 6 ) alkoxy, (C 1 -C 6 )alkanoyl, (C 2 -C 6 )alkanoyloxy, C( ⁇ O) OR a , C( ⁇ O)NR b R c , and NR e R f .
  • R 1 , R 2 or R 3 Another specific value for R 1 , R 2 or R 3 , is (C 1 -C 15 )alkyl, (C 2 -C 15 )alkenyl, (C 2 -C 15 )alkynyl, (C 3 -C 8 )cycloalkyl, (C 3 -C 8 )cycloalkyl(C 1 -C 10 )alkyl, (C 1 -C 15 )alkoxy, (C 1 -C 15 )alkanoyl, or (C 2 -C 10 )alkanoyloxy; wherein said R 2 is optionally substituted with one or more (e.g.
  • substituents independently selected from the group consisting of halo, nitro, cyano, hydroxy, trifluoromethyl, trifluoromethoxy, (C 1 -C 6 )alkoxy, (C 1 -C 6 )alkanoyl, (C 2 -C 6 )alkanoyloxy, C( ⁇ O)OR a , C( ⁇ O)NR b R c , and NR e R f .
  • R 2 is (C 1 -C 15 )alkyl, (C 2 -C 15 )alkenyl, or (C 2 -C 15 )alkynyl, wherein said R 2 is substituted with one or more (e.g. 1, 2, 3, or 4) substituents independently selected from the group consisting of halo, nitro, cyano, hydroxy, trifluoromethyl, trifluoromethoxy, (C 1 -C 6 )alkoxy, (C 1 -C 6 )alkanoyl, (C 2 -C 6 )alkanoyloxy, C( ⁇ O)OR a , C( ⁇ O)NR b R c , and NR e R f .
  • substituents independently selected from the group consisting of halo, nitro, cyano, hydroxy, trifluoromethyl, trifluoromethoxy, (C 1 -C 6 )alkoxy, (C 1 -C 6 )alkanoyl, (C
  • R 2 is (C 1 -C 15 )alkyl, (C 2 -C 15 )alkenyl, or (C 2 -C 15 )alkynyl.
  • R 2 is (C 1 -C 15 )alkyl. Another more specific value for R 2 is (C 1 -C 6 )alkyl. Another more specific value for R 2 is (C 3 -C 6 )alkyl. A preferred value for R 2 is isopropyl or 3-pentyl.
  • FIG. 2 illustrates the synthetic pathway for the synthesis of compound 1.
  • a compound of Formula I can be prepared by deprotection of a corresponding tert-butoxycarbonyl (BOC) protected pyrrolidone using conditions similar to those described in Example 1.
  • BOC tert-butoxycarbonyl
  • Suitable nitrogen protecting groups are well known in the art (See Greene, T. W.; Wutz, P. G. M. “Protecting Groups In Organic Synthesis” second edition, 1991, New York, John Wiley & sons, Inc.).
  • Compounds of Formula I can be prepared from a corresponding compound wherein the hydroxymethyl of Formula I bears a suitable hydroxyl protecting group, by deprotection of the hydroxyl group.
  • a compound of Formula I can be prepared by deprotection of a corresponding tert-butyldimethylsilyl (TBS) protected alcohol using conditions similar to those described in Example 1.
  • TBS tert-butyldimethylsilyl
  • Suitable hydroxyl protecting groups are well known in the art (See Greene, T. W.; Wutz, P. G. M. “Protecting Groups In Organic Synthesis” second edition, 1991, New York, John Wiley & sons, Inc.).
  • Compounds of the invention may also be prepared using procedures similar to those described in Examples 1 and 12, as illustrated in FIGS. 2 - 3 .
  • salts are organic acid addition salts formed with acids which form a physiological acceptable anion, for example, tosylate, methanesulfonate, acetate, citrate, malonate, tartarate, succinate, benzoate, ascorbate, alpha-ketoglutarate, and alpha-glycerophosphate.
  • Suitable inorganic salts may also be formed, including the hydrochloride salt and sulfate, nitrate, bicarbonate, and carbonate salts.
  • salts may be obtained using standard procedures well known in the art, for example by reacting a sufficiently basic compound such as an amine with a suitable acid affording a physiologically acceptable anion.
  • a sufficiently basic compound such as an amine
  • a suitable acid affording a physiologically acceptable anion.
  • Alkali metal (for example, sodium, potassium or lithium) or alkaline earth metal (for example calcium) salts of carboxylic acids can also be made.
  • the compounds of Formula I can be formulated as pharmaceutical compositions and administered to a mammalian host, such as a human patient, in a variety of forms adapted to the chosen route of administration, i.e., orally or parenterally, by intravenous, intramuscular, topical or subcutaneous routes.
  • the present compounds may be systemically administered, e.g., orally, in combination with a pharmaceutically acceptable vehicle such as an inert diluent or an assimilable edible carrier. They may be enclosed in hard or soft shell gelatin capsules, may be compressed into tablets, or may be incorporated directly with the food of the patient's diet.
  • a pharmaceutically acceptable vehicle such as an inert diluent or an assimilable edible carrier.
  • the active compound may be combined with one or more excipients and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like.
  • Such compositions and preparations should contain at least 0.1% of active compound.
  • the percentage of the compositions and preparations may, of course, be varied and may conveniently be between about 2 to about 60% of the weight of a given unit dosage form.
  • the amount of active compound in such therapeutically useful compositions is such that an effective dosage level will be obtained.
  • the tablets, troches, pills, capsules, and the like may also contain the following: binders such as gum tragacanth, acacia, corn starch or gelatin; excipients such as dicalcium phosphate; a disintegrating agent such as corn starch, potato starch, alginic acid and the like; a lubricant such as magnesium stearate; and a sweetening agent such as sucrose, fructose, lactose or aspartame or a flavoring agent such as peppermint, oil of wintergreen, cherry or orange flavoring.
  • a liquid carrier such as a vegetable oil or a polyethylene glycol.
  • any material used in preparing any unit dosage form should be pharmaceutically acceptable and substantially non-toxic in the amounts employed.
  • the active compound may be incorporated into sustained-release preparations and devices.
  • the active compound may also be administered intravenously or intraperitoneally by infusion or injection.
  • Solutions of the active compound or its salts can be prepared in water, optionally mixed with a nontoxic surfactant.
  • Dispersions can also be prepared in glycerol, liquid polyethylene glycols, triacetin, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.
  • the pharmaceutical dosage forms suitable for injection or infusion can include sterile aqueous solutions or dispersions or sterile powders comprising the active ingredient which are adapted for the extemporaneous preparation of sterile injectable or infusible solutions or dispersions, optionally encapsulated in liposomes.
  • the liquid carrier or vehicle can be a solvent or liquid dispersion medium comprising, for example, water, ethanol, a polyol (for example, glycerol, propylene glycol, liquid polyethylene glycols, and the like), vegetable oils, nontoxic glyceryl esters, and suitable mixtures thereof.
  • the proper fluidity can be maintained, for example, by the formation of liposomes, by the maintenance of the required particle size in the case of dispersions or by the use of surfactants.
  • the prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars, buffers or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.
  • Sterile injectable solutions are prepared by incorporating the active compound in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by filter sterilization.
  • the preferred methods of preparation are vacuum drying and the freeze drying techniques, which yield a powder of the active ingredient plus any additional desired ingredient present in the previously sterile-filtered solutions.
  • the present compounds may be applied in pure form, i.e., when they are liquids. However, it will generally be desirable to administer them to the skin as compositions or formulations, in combination with a dermatologically acceptable carrier, which may be a solid or a liquid.
  • Useful solid carriers include finely divided solids such as talc, clay, microcrystalline cellulose, silica, alumina and the like.
  • Useful liquid carriers include water, alcohols or glycols or water-alcohol/glycol blends, in which the present compounds can be dissolved or dispersed at effective levels, optionally with the aid of non-toxic surfactants.
  • Adjuvants such as fragrances and additional antimicrobial agents can be added to optimize the properties for a given use.
  • the resultant liquid compositions can be applied from absorbent pads, used to impregnate bandages and other dressings, or sprayed onto the affected area using pump-type or aerosol sprayers.
  • Thickeners such as synthetic polymers, fatty acids, fatty acid salts and esters, fatty alcohols, modified celluloses or modified mineral materials can also be employed with liquid carriers to form spreadable pastes, gels, ointments, soaps, and the like, for application directly to the skin of the user.
  • Examples of useful dermatological compositions which can be used to deliver the compounds of formula I to the skin are disclosed in Jacquet et al. (U.S. Pat. No. 4,608,392), Geria (U.S. Pat. No. 4,992,478), Smith et al. (U.S. Pat. No. 4,559,157) and Wortzman (U.S. Pat. No. 4,820,508).
  • Useful dosages of the compounds of formula I can be determined by comparing their in vitro activity, and in vivo activity in animal models. Methods for the extrapolation of effective dosages in mice, and other animals, to humans are known to the art; for example, see U.S. Pat. No. 4,938,949.
  • the concentration of the compound(s) of formula I in a liquid composition will be from about 0.1-25 wt-%, preferably from about 0.5-10 wt-%.
  • the concentration in a semi-solid or solid composition such as a gel or a powder will be about 0.1-5 wt-%, preferably about 0.5-2.5 wt-%.
  • Single dosages for injection, infusion or ingestion will generally vary between 50-1500 mg, and may be administered, i.e., 1-3 times daily, to yield levels of about 0.5-50 mg/kg, for adults.
  • the invention includes a pharmaceutical composition
  • a pharmaceutical composition comprising a compound of formula I as described above; or a pharmaceutically acceptable salt thereof, in combination with a pharmaceutically acceptable carrier.
  • Pharmaceutical compositions adapted for oral, topical or parenteral administration, comprising an amount of one or more compounds of formula (I) effective to treat mammalian conditions associated with pathological cellular proliferation, particularly human cancers, such as solid tumors and leukemias, are a preferred embodiment of the invention.
  • simpler phenyl-substituted compounds that retain a hydrogen-bonding network present in the crystal structure, and further contain an isopropyl group that allows strong hydrophobic interaction with PKC residue Leu 254, bind to PKC, but with lower affinity than ILV.
  • This deficiency may be due to the absence of a hydrophobic interaction between the N-methyl group and the pyrrole ring in ILV with PKC.
  • a naphthyl analog designed to restore this interaction exhibited an 8-fold increase in PKC binding affinity.
  • the essential hydrogen bond network, as well as the hydrophobic interactions of PKC residues Pro 241, Leu 251, Leu 254, provide further opportunity for improving affinity and selectivity of novel PKC activators.
  • PKC is an exciting target for cancer therapy.
  • Oncogenes such as src, ras, and sis, elevate phosphatidylinositol turnover; medical transcription of cellular protooncogenes, including myc, and fos, is mediated by PKC; PKC also regulates the activity of the transcriptional activator protein c-jun, and stimulates the multidrug resistance system.
  • PKC also regulates the activity of the transcriptional activator protein c-jun, and stimulates the multidrug resistance system.
  • FIG. 4 is a schematic view of various stages in the calcium signaling cascade. The cascade may be envisioned as occurring in the following six stages illustrated in FIG. 2:
  • Stage I The neuron depolarizes as a result of a convergence of synaptic input, which activates G-protein coupled receptors (e.g. for acetylcholine, GABA, glutamate). Membrane depolarization also opens Ca 2+ channels, causing an influx of Ca 2+ . Diacylglycerol (DAG), arachidonic acid (AA), and inositol triphosphate (IP3) are released by phospholipases, and, along with Ca 2+ , activate protein kinase C (PKC), which is thereby translocated to the plasma membrane. Ca 2+ also activates calmodulin (CaM) kinase. The kinases undergo autophosphorylation, which renders their activity independent of Ca 2+ . PKC and CaM kinase may also inhibit K + and other channels by direct phosphorylation.
  • G-protein coupled receptors e.g. for acetylcholine, GABA, glutamate.
  • IP3 in
  • Stage II Elevated Ca 2+ activates the Ca 2+ -binding protein Cp20, also called calexcitin (CE). Phosphorylation of CE by PKC promotes its translocation to membrane compartments, where it inhibits K + channels, making the membrane more excitable to further depolarizing stimuli. CE also elicits Ca 2+ release from ryanodine receptors (RyR) on the membrane of the endoplasmic reticulum (ER) and possibly synaptic membranes, resulting in amplification of Ca 2+ signals.
  • RyR ryanodine receptors
  • Stage III CE, after phosphorylation by PKC, no longer activates the RyR, but activates Ca 2+ -ATPase at the ER membrane, facilitating the removal of excess Ca 2+ .
  • Stage IV CE and/or Ca 2+ , probably acting indirectly through transcriptional activators, induce new RNA transcription. CE also increases mRNA turnover.
  • Stage V Late genes are transcribed, resulting in increased synthesis of at least 21 different proteins, including RyR. At this stage, retrograde axonal transport is also inhibited by CE; it is believed that this inhibitor may underlie the structural changes in dendritic morphology that are observed after associative learning.
  • Stage VI New RyR receptors and ion channels are synthesized and transported to their respective membranes.
  • a number of the steps of the calcium signaling cascade are defective in AD cells, supporting the notion that calcium homeostasis is involved in the pathophysiology of AD. It is believed that additional defects in the steps of the calcium signaling pathway illustrated in FIG. 2 may exist in AD cells.
  • One important step in the pathway is PKC-mediated phosphorylation of Cp20 (also referred to as CE). Thus, modulation of PKC by activation or deactivation presents a viable treatment for AD.
  • the 2-pyrrolidone PKC activators of Kozikowski et al. may be employed in treating conditions characterized by the pathological proliferation of mammalian cells (e.g. cancer).
  • mammalian cells e.g. cancer
  • Kozikowski et al. concluded that the 2-pyrrolidones were useful as isozyme selective, non-tumor promoting activators of PKC that cause downregulation and, in the context of cancer treatment, cause initiation of cancer cell death through apoptosis.
  • selective cancer cell killing may be achieved either through the targeting of those isozymes found to be overexpressed in the cancer cells, or through the synergistic interaction of a cytotoxic drug like 1-D-arabinofuranosylcytosine with an appropriate PKC-based signaling interceptor.
  • PKC can be activated by phorbol esters which significantly increase the relative amount of non-amyloidogenic soluble APP (sAPP) secreted.
  • sAPP non-amyloidogenic soluble APP
  • phorbol ester does not appear to result in a direct phosphorylation of the APP molecule, however. Irrespective of the precise site of action, phorbol-induced PKC activation results in an enhanced or favored ⁇ -secretase, non-amyloidogenic pathway. Potentially then, PKC activation could be an attractive approach to influence the production of non deleterious and even beneficial sAPP and at the same time reduce the relative amount of A ⁇ peptides.
  • Phorbol esters may not be suitable compounds for eventual drug development because of their tumorigenic activity.
  • Activation of PKC is a multi-step process. Independent of the stimulus, PKC activation involves first, and almost universally, the movement of the enzyme from the cytosol to specific binding domains at cell membranes. This process is termed translocation. For the classic isozymes, an intracellular calcium elevation triggers this event. A subsequent interaction with diacylglycerol (DAG) facilitates penetration into the membrane and complete activation. DAG, although not calcium, is also required to activate the novel isozymes. Translocation does not measure the enzymatic activity per se, i.e. the capacity to phosphorylate a substrate, but it is a prerequisite. Therefore, translocation is a good indicator of the presence of the enzyme in its active form and conformation, thus “biologically ready” to exert its actions. Translocation is commonly and easily measured in the laboratory (Western blots).
  • Preferred compounds for anti-carrier effects are those with high affinities of ⁇ - and ⁇ -isozymes. Results are shown in Table 1. (See the key that follows) TABLE 1 whole brain PKC ⁇ -PKC ⁇ -PKC ⁇ -PKC ⁇ -PKC Cpd.
  • a compound is specific to a PKC isozyme or group of isozymes if the compound has an affinity for the target isozyme(s) that is higher than the affinity for non-target isozyme or group of isozymes, preferably significantly higher, and if the affinity for the non-target isozyme(s) is as low as possible.
  • one group of preferred compounds are those in which affinity for ⁇ , ⁇ and ⁇ isozymes is the greatest (i.e. those having the lowest K i ) relative to other isozymes such as the ⁇ - and ⁇ -isozymes shown in Table 1.
  • Preferred compounds for specific disease have specificity for particular isozymes.
  • Preferred compounds for neurological treatment have affinities for ⁇ , ⁇ and ⁇ isozymes that are significantly higher than for other non CNS-specific isozymes. Affinity for ⁇ isozyme is particularly preferred. For prostate cancer, specificity to the ⁇ isoform is preferred. For other disorders, specificity to other isozymes is preferred.
  • the present inventors have studied benzolactams and flexible 2-pyrrolidones as activators of protein kinase (PKC). Alterations in PKC, as well as alterations in calcium (Ca 2+ ) regulation and potassium (K + ) channels are included among alterations in fibroblasts in Alzheimer's disease (AD) patients. Since PKC is known to regulate ion channels, the present inventors have studied K + channel activity in fibroblasts from AD patients in the presence of (2S, 5S)-8-(1-decynyl)benzolactam (BL), a novel activator of PKC with improved selectivity for the ⁇ , ⁇ , and ⁇ isozymes.
  • PKC protein kinase
  • AD Alzheimer's disease
  • animal neuronal cells permitted the identification of a number of cellular/molecular alterations that may be the reflection of comparable processes in the AD brain and thus, of pathophysiological relevance (Baker et al., 1988; Scott, 1993; Huang, 1994; Scheuner et al., 1996; Etcheberrigaray & Alkon, 1997; Gasparini et al., 1997). Alterations of potassium channel function have been identified in fibroblasts (Etcheberrigaray et al., 1993) and in blood cells (Bondy et al., 1996) obtained from AD patients.
  • ⁇ -amyloid widely accepted as a major player in AD pathophysiology (Gandy & Greengard, 1994; Selkoe, 1994; Yankner, 1996), was capable of inducing an AD-like K + channel alteration in control fibroblasts (Etcheberrigaray et al., 1994). Similar or comparable effects of ⁇ -amyloid on K + channels have been reported in neurons from laboratory animals (Good et al., 1996; also for a review see Fraser et al., 1997).
  • TEA-induced [Ca 2+ ] elevation was used because it has been shown to depend on functional 113pS K + channels that are susceptible to TEA blockade (Etcheberrigaray et al., 1993, 1994; Hirashima et al., 1996).
  • TEA-induced [Ca 2+ ] elevations and K + channel activity are primarily observed in fibroblasts from control individuals while being virtually absent in fibroblasts from AD patients (Etcheberrigaray et al., 1993; Hirashima et al., 1996).
  • the present inventors have also observed that activation of protein kinase C favors the ⁇ -secretase processing of the Alzheimer's disease (AD) amyloid precursor protein (APP), resulting in the generation of non-amyloidogenic soluble APP (sAPP). Consequently, the relative secretion of amyloidogenic A ⁇ 1-40 and A ⁇ 1-42(3) is reduced. This is particularly relevant since fibroblasts and other cells expressing APP and presenilin AD mutations secrete increased amounts of total A ⁇ and/or increased ratios of A ⁇ 1-42(3) /A ⁇ 1-40 . Interestingly, PKC defects have been found in AD brain ( ⁇ , ⁇ and ⁇ isozymes) and in fibroblasts ( ⁇ -isozyme) from AD patients.
  • a novel PKC activator (benzolactam, BL) with improved selectivity for the ⁇ , ⁇ and ⁇ isozymes to enhance sAPP secretion over basal levels.
  • the sAPP secretion in BL-treated AD cells was also slightly higher compared to control BL-treated fibroblasts, which only showed significant increases of sAPP secretion after treatment with 10 ⁇ M BL.
  • Staurosporine (a PKC inhibitor) eliminated the effects of BL in both control and AD fibroblasts.
  • BL causes approximately a 3-fold sAPP secretion in PC12 cells.
  • the use of a novel and possibly non-tumorigenic PKC activator may prove useful to favor non-amyloidogenic APP processing and is, therefore, of potential therapeutic value.
  • PKC activators that selectively act on ⁇ , ⁇ and ⁇ isozymes while having a lesser affect on other isozymes are of particular interest.
  • the PKC ⁇ is the most abundant isozyme in the brain and PKC ⁇ is the most specific isozyme for the brain.
  • Such activators of ⁇ , ⁇ and ⁇ isozymes have the potential to significantly affect PKC in the brain, while having minimal effects on the rest of the body.
  • preferred PKC activators are those that have higher selectivity toward ⁇ , ⁇ and ⁇ isozymes and a lower activity with respect to at least one of the other isozymes. More preferred compounds would have a significantly larger effect on ⁇ , ⁇ and ⁇ isozymes as compared to other isozymes.
  • APP amyloid precursor protein
  • Mass spectra were obtained in electron impact ionization mode at 70 eV. Optical rotations were measured at room temperature.
  • ⁇ D ⁇ 83.1 (c 0.50 in CHCl 3 );
  • Reagents and conditions in FIG. 3 (a) NBS, CH 3 CN, rt, 1 h, 95%. (b) lauroyl chloride, pyridine, DMAP (cat.), CH 2 Cl 2 , rt, overnight, 90%. (c) 48% HF, CH3CN, rt, overnight, 95%. (d) 1-heptyne, PdCl 2 (PPh 3 ) 2 (5% mol), CuI (10% mol), n-Bu 4 NI, Et 3 N, DMF, 80° C., 2 days, 80%. (e) Tf 2 O, 2,6-lutidine, CH 2 Cl 2 , ⁇ 78° C. to ⁇ 40° C., 90%;%. (g) 1-decyne, PdCl 2 (PPh 3 ) 2 (5% mol), CuI (10% mol), n-Bu 4 NI, Et 3 N, DMF, 80° C., 2 days, 80%.
  • Binding was determined in an incubation volume of 250 ⁇ l, containing [ 3 H]PDBu, PKC, 0.05 M Tris-Cl, pH 7.4, 100 ⁇ g/ml phosphatidylserine, various concentrations of the ligand being assayed for competition of [ 3 H]PDBu binding, and bovine x-globulin at 1 mg/ml.
  • PKCs ⁇ , ⁇ , ⁇
  • CaCl 2 was included at a concentration of 0.1 mM.
  • Liposomes of phosphatidylserine were prepared as follows. The phosphatidylserine solutions were dried down from CHCl 3 under a stream of N 2 .
  • the precipitate was recovered by centrifugation at 12,000 rpm in a Beckman microcentrifuge at 4° C. A 100 ⁇ l aliquot of the supernatant was removed and its radioactivity determined in a Wallac liquid scintillation counter, Model 1409. This value provided a measure of the concentration of unbound [ 3 H]PDBu. The remainder of the supernatant was removed by aspiration and blotting with absorbent tissue. The tip of the centrifuge tube was cut off and the radioactivity in the pellet measured to determine total bound [ 3 H]PDBu. Non-specific binding of [ 3 H]PDBu was measured in the presence of 30 ⁇ M non-radioactive PDBu and used to determine an apparent partition coefficient for [ 3 H]PDBu under these assay conditions.
  • Specific binding represents the difference between total binding and the non-specific binding, where the latter value was calculated from the partition coefficient and the measured free [ 3 H]PDBu concentration in each tube.
  • triplicate determinations were performed at each concentration of competing ligand.
  • three experiments were performed to confirm the reproducibility of the measured K i .
  • PKC isozymes are expressed in the baculovirus system and partially purified as has been previously described.(Kazanietz, et al., Mol. Pharmacol. 44:298-307 (1993); Areces, et al., J. Biol. Chem. 269:19553-19558 (1994); Caloca, et al., J. Biol. Chem. 272:26488-26496 (1997)). Briefly, 1 liter of approximately 2 ⁇ 106 Sf9 cells/ml in spinner flasks was infected with the recombinant viruses at a multiplicity of infection of 10. After 60-72 hr, the cells were centrifuged (1000 rpm, 10 min) and washed twice with phosphate buffered saline, and the pelleted cells kept at ⁇ 70° C. until used.
  • the cell pellet was resuspended in 50 mnl of a homogenization buffer of the following composition; 20 mM Tris-Cl, pH 8.0, 5 mM EDTA, 5 mM EGTA, 0.3% (v/v) 2-mercaptoethanol, 10 mM benzamidine, 50 ⁇ g/ml phenylmethylsulfonyl fluoride, and 250 ⁇ g/ml leupeptin.
  • the cells were disrupted in a Potter-Elvehjem homogenizer at 4° C., and the homogenate is centrifuged at 100,000 ⁇ g for 60 min.
  • the supernatant was adjusted to pH 8.0 and loaded in a TSK-GEL DEAE-5PW column (15 cm ⁇ 2 cm; Tosohaas, Philadelphia, Pa.) that was equilibrated with 20 mM Tris-Cl, pH 7.5, 2 mM EDTA, 0.3% 2-mercaptoethanol, 10 mM benzamidine.
  • the colunm was eluted with a 375 ml linear gradient of NaCl (0-400 mM) in the equilibration buffer at a flow rate of 2.5 ml/min. Fractions were dialyzed against 20 mM Tris-Cl, pH 7.5, 1 mM dithiothreitol, 1 mM EDTA, 50% glycerol, before storage at ⁇ 70° C.
  • compounds of the invention are useful for treating diseases or conditions wherein PKC activity is implicated and wherein activation of PKC is desirable.
  • compounds of the invention may be useful for treating a disease or condition characterized by the pathological proliferation of mammalian cells, such as for example, human cancers, such as prostate cancer, solid tumors and leukemias.
  • Compounds of the invention may also be useful for treating autoimmune diseases, neurological disorders such as Alzheimer's disease, and inflammation.
  • the invention includes a method comprising modulating PKC in a mammal by administering to said mammal a pharmaceutically effective dose of a compound of formula I; or a pharmaceutically acceptable salt thereof.
  • the invention also provides a method comprising treating a condition characterized by the pathological proliferation of mammalian cells by administering to a mammal afflicted with such a condition, an effective amount of a compound of formula I; or a pharmaceutically acceptable salt thereof.
  • Examples 14 and 15 utilize methods which have been previously described with respect to benzolactams. Ibarreta D, Duchen M, Ma D, Qiao L, Kozikowski A P, Etcheberrigaray R., “Benzolactam (BL) enhances sAPP secretion in fibroblasts and in PC 12 cells.” Neuroreport 1999 Apr 6;10(5):1035-40; Bhagavan S, Ibarreta D, Ma D, Kozikowski A P, Etcheberrigaray R., “Restoration of TEA-induced calcium responses in fibroblasts from Alzheimer's disease patients by a PKC activator.” Neurobiol Dis 1998 Sep;5(3):177-87. These results have now been found extended remarkably to certain rigid pyrrolidones.
  • Human fibroblasts were grown to confluence in 100 mm tissue culture dishes. On the day of the experiment, cells were rinsed twice with serum-free Dulbecco's modified Eagle's medium (DMEM) and incubated in the same medium for 2 hours. Cells were treated with the drugs above, or DMSO vehicle as control, for 15 min. The cells were then rinsed twice with ice-cold phosphate buffered saline (PBS), scraped in PBS, and collected by centrifugation.
  • DMEM serum-free Dulbecco's modified Eagle's medium
  • PBS ice-cold phosphate buffered saline
  • the pellets were suspended in homogenization buffer containing: 20 mM Tris-HCl, pH 7.5, 2 mM EDTA, 2 mM EGTA, 5 mM DTT, 0.32 M sucrose, 2 mM PMSF, 25 g/ml aprotinin, and 20 g/ml leupeptin. Homogenates were immersed in liquid nitrogen thawed, and centrifuged at 12,000 g for 20 min, and supernatants were used as the cytosolic fraction (C).
  • the pellets were homogenized in the same buffer containing 1.0% Triton X-100, incubated on ice for 45 min, and centrifuged at 12,000 g for 30 min and the supernatants were used as the membrane fractions (M). After protein determination, equal amounts of proteins (approx. 20 g) were mixed with electrophoresis buffer, boiled for 5 min, and separated on 8% SDS-PAGE, and electrophoretically transferred to PVDF membranes. The membranes were processed by Western blotting using a specific antibody for PKC isozyme.
  • the protein pellets were washed with ice-cold acetone and centrifuged for 30 minutes at 20,000 g for 30 minutes and dried overnight. Pellets were dissolved in electrophoresis buffer and proteins corresponding to the same concentration of protein were separated by SDS-PAGE and transferred to PVDF membranes according to the manufacturer's instructions. After electrophoresis, the membranes were blocked with Blotto and incubated overnight with a monoclonal antibody 6E10 (Synetek) that recognizes the APP ⁇ .
  • a monoclonal antibody 6E10 Synetek
  • alkaline phosphatase anti-mouse IgG Jackson's Laboratories
  • the membranes were developed using an alkaline phosphatase kit (BioRad) as per the manufacturer's instructions.
  • Compound (1) at concentration ranges between 10-20 ⁇ M, increased the secretion of soluble APP ⁇ (sAPP ⁇ ) significantly.
  • PKC ⁇ and PKC ⁇ are the isozymes that mediate phorbol ester-induced apoptosis in LNCaP cells (references 13c-e).
  • a model that has been used to assess isozyme-specificity is the overexpression of specific PKCs by an adenoviral system. Infection of LNCaP cells with adenoviruses for PKCs leads to a high expression of the corresponding PKC isozyme, as determined by Western-blot analysis, kinase activity, or [ 3 H]PDBu binding (reference 13e).

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US8785648B1 (en) 2010-08-10 2014-07-22 The Regents Of The University Of California PKC-epsilon inhibitors
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